The present invention relates generally to risk-assessment of suspected genotoxins, evaluation of novel chemotherapeutic agents, and novel chemotherapeutic methods for cancer management.
Cancer arises when a normal cell undergoes neoplastic transformation and becomes a malignant cell. Transformed (malignant) cells escape normal physiologic controls specifying cell phenotype and restraining cell proliferation. Transformed cells in an individual""s body thus proliferate, forming a tumor (also referred to as a neoplasm). When a neoplasm is found, the clinical objective is to destroy malignant cells selectively while mitigating any harm caused to normal cells in the individual undergoing treatment. Currently, three major approaches are followed for the clinical management of cancer in humans and other animals. Surgical resection of solid tumors, malignant nodules and or entire organs may be appropriate for certain types of neoplasia. For other types, e.g., those manifested as soluble (ascites) tumors, hematopoeitic malignancies such as leukemia, or where metastasis of a primary tumor to another site in the body is suspected, radiation or chemotherapy may be appropriate. Either of these techniques also is commonly used as an adjunct to surgery. Harrison""s Principles of Internal Medicine, Part 11 Hematology and Oncology, Ch. 296, 297 and 300-308 (12th ed. 1991).
Chemotherapy is based on the use of drugs that are selectively toxic (cytotoxic) to cancer cells. Id. at Ch. 301. Several general classes of chemotherapeutic drugs have been developed, including drugs that interfere with nucleic acid synthesis, protein synthesis, and other vital metabolic processes. These generally are referred to as antimetabolite drugs. Other classes of chemotherapeutic drugs inflict damage on cellular DNA. Drugs of these classes generally are referred to as genotoxic. Two widely used genotoxic anticancer drugs that have been shown to damage cellular DNA by producing crosslinks therein are cisplatin [cis-diamminedichloroplatinum(II)] and carboplatin [diammine(1,1-cyclobutanedicarboxylato)platinum(II)]. Bruhn et al. (1990), 38 Prog. Inorg. Chem. 477, Burnouf et al. (1987), 84 Proc. Natl. Acad. Sci. USA 3758, Sorenson and Eastman (1987), 48 Cancer Res. 4484 and 6703, Pinto and Lippard (1985), 82 Proc. Natl. Acad. Sci., USA 4616, Lim and Martini (1984), 38 J. Inorg. Nucl. Chem. 119, Lee and Martin (1976), 17 Inorg. Chim. Acta 105, Harder and Rosenberg (1970), 6 Int. J. Cancer 207, Howle and Gale (1970), 19 Biochem. Pharmacol 2757. Cisplatin and/or carboplatin currently are used in the treatment of selected, diverse neoplasms of epithelial and mesenchymal origin, including carcinomas and sarcomas of the respiratory, gastrointestinal and reproductive tracts, of the central nervous system, and of squamous origin in the head and neck. Harrison""s Principles of Internal Medicine (12th ed. 1991) at Ch. 301. Cisplatin currently is preferred for the management of testicular carcinoma, and in many instances produces a lasting remission. Loehrer and Einhorn (1984), 100 Ann. Int. Med. 704. Susceptibility of an individual neoplasm to a desired chemotherapeutic drug or combination thereof often, however, can be accurately assessed only after a trial period of treatment. The time invested in an unsuccessful trial period poses a significant risk in the clinical management of aggressive malignancies.
The repair of damage to cellular DNA is an important biological process carried out by a cell""s enzymatic DNA repair machinery. Unrepaired lesions in a cell""s genome can impede DNA replication, impair the replication fidelity of newly synthesized DNA or hinder the expression of genes needed for cell survival. Thus, genotoxic drugs generally are considered more toxic to actively dividing cells that engage in DNA synthesis than to quiescent, nondividing cells. Indeed, cells carrying a genetic defect in one or more elements of the enzymatic DNA repair machinery are extremely sensitive to cisplatin. Fraval et al. (1978), 51 Mutat. Res. 121, Beck and Brubaker (1973), 116 J. Bacteriol 1247. Normal cells of many body tissues, however, are quiescent and commit infrequently to re-enter the cell cycle and divide. Greater time between rounds of cell division generally is afforded for the repair of DNA damage in normal cells inflected by chemotherapeutic genotoxins. As a result, some selectivity is achieved for the killing of cancer cells. Many treatment regimes reflect attempts to improve selectivity for cancer cells by coadministering chemotherapeutic drugs belonging to two or more of these general classes.
In some tissues, however, normal cells divide continuously. Thus, skin, hair follicles, buccal mucosa and other tissues of the gut lining, sperm and blood-forming tissues of the bone marrow remain vulnerable to the action of genotoxic drugs, including cisplatin. These and other classes of chemotherapeutic drugs can also cause severe adverse side effects in drug-sensitive organs, such as the liver and kidneys. These and other adverse side effects seriously constrain the dosage levels and lengths of treatment regimens that can be prescribed for individuals in need of cancer chemotherapy. Harrison""s Principles of Internal Medicine (12th ed. 1991) at Ch. 301. See also Jones et al. (1985), 52 Lab. Invest. 363-374 and Loehrer and Einhorn (1984), 100 Ann. Int. Med. 704-714. Such constraints can prejudice the effectiveness of clinical treatment. For example, the drug or drug combination administered must contact and affect cancer cells at times appropriate to impair cell survival. Genotoxic drugs are most effective for killing cancer cells that are actively dividing when chemotherapeutic treatment is applied. Conversely, such drugs are relatively ineffective for the treatment of slow growing neoplasms. Carcinoma cells of the breast, lung and colorectal tissues, for example, typically double as slowly as once every 100 days. Harrison""s Principles of Internal Medicine (12th ed. 1991) at Table 301-1. Such slowly growing neoplasms present difficult chemotherapeutic targets.
Moreover, cancer cells can acquire resistance to genotoxic drugs through diminished uptake or other changes in drug metabolism, such as those that occur upon drug-induced gene amplification or expression of a cellular gene for multiple drug resistance (MDR). Harrison""s Principles of Internal Medicine (12th ed. 1991) at Ch. 301. Resistance to genotoxic drugs also can be acquired by activation or enhanced expression of enzymes in the cancer cell""s enzymatic DNA repair machinery. Therapies that employ combinations of drugs, or drugs and radiation, attempt to overcome these limitations. The pharmacokinetic profile of each chemotherapeutic drug in such a combinatorial regime, however, will differ. In particular, permeability of neoplastic tissue for each drug will be different. Thus, it can be difficult to achieve genotoxically effective concentrations of multiple chemotherapeutic drugs in target tissues.
Needs remain for additional chemotherapeutic drugs with improved selectivity for destroying transformed cells in situ, without significantly impairing viability of untransformed cells. Needs remain also for enhancing effectiveness of chemotherapeutic drugs, such that satisfactory cell killing can be achieved with lower doses thereof than are currently needed. Thus, needs remain for improved, more accurate methods of testing whether a given chemotherapeutic drug will be effective for killing a particular colony of transformed cells in situ. Poignant needs remain for chemotherapeutic drugs with improved selectivity for destroying transformed cells. Particularly poignant needs remain for ways to render transformed cells selectively more vulnerable to killing through chemotherapy.
It is an object of this invention to provide a method for assessing whether a suspected genotoxic agent forms lesions in DNA that are bound (recognized) by a DNA structure specific recognition protein (SSRP). Thus, it is an object of this invention to provide an in vitro assay for predicting whether a suspected genotoxic agent forms persistent genomic lesions in eukaryotic cellular DNA.
Another object of this invention is to provide a method for assessing whether a eukaryotic cell contains a DNA structure specific recognition protein that binds to DNA lesions formed by a genotoxic agent. Thus, it is an object of this invention to provide a method for predicting susceptibility of a eukaryotic cell to killing by a genotoxic agent.
Yet another object of this invention is to provide a method of screening new genotoxic drug candidates for the ability to form DNA lesions that are bound by a DNA structure specific recognition protein. Thus, it is an object of this invention to provide a screening method for the rational design of new genotoxic drugs that form persistent genomic lesions in eukaryotic cells. Accordingly, it is an object of this invention to provide new genotoxic drugs identified from the screening method described herein.
Still another object of this invention to provide a method of causing a eukaryotic cell to express a DNA structure specific recognition protein encoded by a heterologous nucleic acid. Thus, it is an object of this invention to provide a method for enhancing persistence of DNA lesions in the genome of eukaryotic cells. The objects of this invention accordingly include providing a method for sensitizing eukaryotic cells to killing by a genotoxic agent. A further object of this invention therefore includes providing an improved method for killing eukaryotic cells, based on rendering the cells sensitive to a genotoxic agent by causing said cells to express a DNA structure specific recognition protein, and then exposing the cells to the genotoxic agent.
These and other objects, along with advantages and features of the invention disclosed herein, will be apparent from the description, drawings and claims that follow.
The invention described herein rests on the discovery that eukaryotic cells contain one or more intracellular structure specific recognition proteins (SSRPs) that bind to sequence-independent structural motifs in cellular DNA produced by the binding thereto of genotoxic agents. Genotoxic agents or genotoxins bind to or otherwise physically or chemically interact with cellular DNA, causing injury thereto. A site of injury (a lesion) in cellular DNA is referred to herein as a genomic lesion. DNA lesions can include disruptions of the nucleotide sequence, nucleotide basepairing, or distortions of the structure of the DNA double helix. Structural distortion lesions produce three-dimensional DNA structural motifs (e.g., bends, kinks, unwinding, overwinding, non-B helical forms such as A- or Z-DNA, junctions between different helical forms, stem-loop structures, cruciforms, local melting, crossover junctions and the like). Genomic lesions in cellular DNA that are not repaired before the cell commits itself to the cycle of cell division contribute to cell death. Thus, one determinant of a genotoxic agent""s cytotoxicity (propensity for contributing to cell death) is the resistance of genomic lesions formed therefrom to cellular repair. Genotoxic agents that form persistent genomic lesions, e.g., lesions that remain in the genome at least until the cell commits to the cell cycle, generally are more effective cytotoxins than agents that form transient, easily repaired genomic lesions. Hence, genotoxic agents that form persistent genomic lesions are preferred for use as chemotherapeutic agents in the clinical management of cancer.
The invention rests more precisely on the discovery, recounted in U.S. Pat. No. 5,359,047 (incorporated herein by reference), that eukaryotic cells contain one or more SSRPs that bind to 1,2-dinucleotide intrastrand adducts of genotoxic metal coordination compounds currently used as chemotherapeutic agents in the clinical management of cancer. Such genotoxic metal coordination compounds include noble metal compounds, such as platinum(II) and platinum(IV) compounds. Typically, the compounds comprise a platinum atom linked to a pair of cis-configured substitutionally labile moieties and a pair of cis-configured electron donor moieties. Binding of the noble metal coordination compounds to nucleic acids occurs upon substitution of the cis-configured labile moieties with atoms of the nucleotide bases, usually adenosine (A) or guanine (G) residues. This produces a crosslink, bridged by the noble metal atom (e.g., platinum) between two vicinal, adjacent or paired nucleotide bases. Platinum-bridged crosslinks between adjacent adenosine and/or guanine residues within a single nucleotide strand (1,2-intrastrand dinucleotide adducts or lesions) of double stranded DNA are abbreviated herein as 1,2-d(A{circumflex over ( )}G) and 1,2-d(G{circumflex over ( )}G) lesions. The class of genotoxic noble metal coordination compounds that form SSRP-recognized genomic lesions includes cisplatin (cis-diamminedichloroplatinum(II) or cis-DDP), carboplatin (diammine(1,1-cyclobutane-dicarboxylato)platinum(II), cis-diamminetetrachloroplatinum(IV), iproplatin (CHIP), DACCP, malonatoplatin, cis-dichloro(ethylenediamine)platinum(II), cis-dichloro(1,2-diaminocyclohexyl)platinum(II), and the like. For convenience, SSRP recognized 1,2-intrastrand dinucleotide adducts formed by any member of this class are referred to herein as cisplatin-type lesions (or adducts).
SSRPs have been shown to bind to the 1,2-d(A{circumflex over ( )}G) or 1,2-d(G{circumflex over ( )}G) intrastrand DNA adducts of cisplatin irrespective of the 5xe2x80x2 or 3xe2x80x2 orientation of the lesion site and irrespective of the nucleotide sequence adjacent to or comprising the lesion site. Hence, SSRP binding is understood to be sequence-independent, in contrast to the binding properties of other, known nucleic acid binding proteins. SSRP binding to the 1,2-intrastrand dinucleotide adduct (lesion) of a cisplatin-type genotoxic agent results in the formation of a lesioned DNA/SSRP complex. This complex can be detected visually using techniques described in U.S. Pat. No. 5,359,047, including modified Western (Southwestern) blotting and electrophoretic mobility shift analysis (EMSA, also known as bandshift analysis).
SSRPs thus far reported to bind to 1,2-intrastrand cisplatin-type lesions in DNA comprise at least one structural domain generally referred to as an HMG domain. Exemplary, preferred SSRP HMG domains include the HMG domains of human and Drosophila SSRP1, having the sequences set forth, respectively, in amino acid residues 539-614 of Seq. ID No. 2 and residues 547-620 of Seq. ID No. 6. Other useful SSRP HMG domains are encoded by nucleic acids that hybridize specifically, at least under low stringency hybridization conditions such as described in U.S. Pat. No. 5,359,047, to nucleic acid encoding the HMG domain of human or Drosophila SSRP1. SSRPs comprising such HMG domains and occurring in non-human or non-Drosophila eukaryotes are considered homologs of human or Drosophila SSRP1. SSRP-encoding homologous nucleic acids have been detected in diverse eukaryotes, including arthropods (represented by the fruitfly Drosophila melanogaster) and vertebrates including mammals (e.g., human, chimpanzee, monkey, elephant, pig, dog, rabbit, mouse and opossum), aves (e.g., chicken) and fish. It is deduced that homologs of the human and/or Drosophila SSRP occur in numerous eukaryotes, including at least arthropods and vertebrates. A mouse protein comprising an SSRP HMG domain and considered to be a homolog of human SSRP1 has been referred in the literature as T160. SSRP variants occurring within a given eukaryotic species (e.g., humans) that are encoded by nucleic acids comprising sequences similar but not identical to, e.g., residues 539-614 of Seq ID No. 2 (human SSRP1), are understood to be polymorphic or allelic SSRP1 variants. Homologous and polymorphic SSRP1 variants also are useful in the invention described herein.
Proteins comprising still other useful SSRP HMG domains can be identified empirically, based upon their ability to form detectable cisplatin-lesioned DNA/protein complexes. Such other useful SSRP HMG domains need not be encoded by nucleic acid that hybridizes specifically to nucleic acid encoding the HMG domain of human or Drosophila SSRP1. At least one such empirically identified, useful SSRP is fractional yeast SSRP (fySSRP), Seq. ID No. 8. This SSRP has been referred to in publications as IXR-1 (intrastrand crosslink recognition protein 1). Additional useful SSRP HMG domains occur in such known HMG proteins as HMG-1, HMG-2, UBF, LEF-1, SRY, mtTFA, ABF2 and the like. These and other known HMG domain SSRPs have been isolated, variously, from diverse eukaryotes, including human, rodent, Xenopus, Drosophila and yeast.
The consequence of SSRP binding to a genomic lesion is that the sterically large SSRP (or a fragment thereof comprising an HMG domain) becomes localized in the immediate vicinity of the genomic lesion. The SSRP is large enough to sterically obscure (cover) a region of cellular DNA extending from the lesion site in either the 5xe2x80x2 and 3xe2x80x2 direction for at least about five base pairs, preferably at least about eight base pairs, more preferably at least about twelve base pairs. As a result, lesion-bound SSRP shields the genomic lesion from repair by the cell""s enzymatic DNA repair machinery. SSRP-shielded lesions persist in the genome longer than unshielded lesions. SSRP-shielded lesions accordingly are more effective for prejudicing the fidelity of DNA replication, hindering the expression of genes relevant to cell survival, and otherwise contributing to disarray of the cell""s nuclear architecture. One or more of the foregoing can contribute to cell death, e.g., by triggering apoptosis.
Certain HMG domain proteins useful herein as SSRPs have been characterized in the literature as transcription factors that control or modulate the expression of one or more cellular genes, including genes that are relevant to cell metabolism or cell secretory function. One such transcription factor is upstream binding factor (UBF), which controls the expression of ribosomal RNA genes and thus is pivotal to the function of the cell""s protein synthesis machinery. It is thought that cisplatin-type lesions to which such transcription factors bind as SSRPs mimic or resemble the factor""s natural genomic binding site. Binding of such transcription factors to cisplatin-type genomic lesions in effect sequesters the transcription factors at sites other than the natural genomic binding site. Titration of the transcription factors away from their natural genomic binding sites contributes to dysregulation of the controlled genes and therefore contributes to disarray of cellular processes and functions directed by the products (generally proteins, e.g., enzymes) of the controlled genes. For example, sequestration or xe2x80x9chijackingxe2x80x9d of the HMG domain transcription factor UBF by cisplatin-type lesions contributes to disarray of cellular protein synthesis, a process needed for cell survival.
The invention described herein accordingly features, in one aspect, a method for predicting cytotoxicity of an agent that binds to DNA (a genotoxic agent or genotoxin). In this method, a sample of double-stranded DNA bearing a lesion formed by the genotoxic agent is contacted with a DNA structure-specific recognition protein, such that a lesioned DNA/SSRP complex forms. This complex is detected or visualized, and optionally quantitated e.g., relative to a standard genotoxic agent known to form a DNA lesion bound by the SSRP. Capacity of the genotoxic agent to form SSRP-shielded DNA lesions in vitro is considered reasonably predictive of competence of the agent to form persistent genomic lesions in cellular DNA, rather than transient, easily repaired lesions.
In another aspect, the invention features a method for assessing cytotoxicity of an agent that inflicts genomic lesions on cellular DNA. That is, the invention features a method for predicting susceptibility of eukaryotic cells to the cytotoxic effects of a genotoxin. In this method, a sample comprising eukaryotic cells is treated so as to release intracellular proteins. The released intracellular proteins are assessed for the presence of one or more DNA structure-specific recognition proteins that bind to DNA lesioned by the genotoxin. Thus, released intracellular proteins are contacted with probe DNA comprising at least one lesion formed by the genotoxin, such that a lesioned probe DNA/cellular SSRP complex forms. This complex is detected or visualized, and optionally quantitated e.g., relative to a standard SSRP known to bind DNA lesions formed by the genotoxic agent. Presence within the eukaryotic cells of one or more SSRPs that bind to the lesioned probe DNA is considered reasonably predictive of formation of persistent genomic lesions in cellular DNA. Accordingly, the presence and amount of SSRPs within the eukaryotic cells can be used to confirm whether a desired genotoxic agent will be cytotoxic to the cells, as well as to assist in the calculation of the dose of genotoxic agent needed to produce the desired degree or rapidity of cell killing.
In yet another aspect, the invention features a method for identifying novel cytotoxic agents that bind to DNA to form genomic lesions. That is, the invention features a screening method for assessing new, genotoxic drug candidates for the ability to form SSRP-recognizable and thus persistent genomic lesions. This method involves contacting a sample of DNA, optionally comprising a detectable moiety, with one or more candidate cytotoxic agents, then incubating the DNA with the candidate under conditions sufficient for DNA binding of genotoxic agents. The DNA bearing a genomic lesion formed by a candidate genotoxin is separated from the incubation mixture comprising unlesioned DNA and unbound candidate. Successfully lesioned DNA is contacted an SSRP under conditions sufficient for the formation of a lesioned DNA/SSRP complex, which is thereupon detected. Optionally, SSRP can be used as an affinity separation agent to isolate successfully lesioned DNA from the incubation mixture. This rational drug screening method can be automated for high-throughput screening of numerous candidate compounds. It is suitable for screening random libraries of compounds, e.g., libraries produced by random or directed combinatorial synthesis of inorganic, organic or biological compounds. The invention accordingly encompasses new cytotoxic agents identified according to the present screening method.
Suitable methods for detecting lesioned DNA/SSRP complexes formed in the above aspects of the present invention include EHSA and Southwestern blotting, both generally according to U.S. Pat. No. 5,359,047. In these and other methods described herein, detection can optionally be facilitated through the use of lesioned probe DNA. Probe DNA is a fragment (e.g., a restriction fragment) of naturally occurring or recombinant DNA, or is a synthetically constructed DNA, of a size suitable for use in standard analytical procedures. For example, the probe DNA is at least about 60 basepairs (bp), preferably at least about 80 bp, more preferably at least about 100 bp in length. Lesioned probe DNA contains at least one structural motif (lesion) produced by the binding thereto of a genotoxic agent. Optionally, the probe DNA also comprises a detectable moiety, such as a radioisotope, chromophore, fluorophore, hapten or other high affinity ligand (e.g., biotin). Other methods for detecting lesioned DNA/SSRP complexes, optionally involving the use of a suitable probe DNA, include nitrocellulose filter retention assay and excinuclease protection assay, both described herein. The nitrocellulose filter retention assay is based upon the selective retention or filter-binding of proteins such as SSRPs. Lesioned probe DNA binds to the SSRP and thus is retained by the filter, whereas unlesioned probe DNA (or probe DNA bearing an unrecognized lesion) flows through or is not retained by the filter. If desired, the filter can be blocked or treated to reduce nonspecific retention. Nitrocellulose filter retention assays can be carried out, e.g., using a standard dot blotting apparatus. The selective retention principle of the nitrocellulose filter retention assay can be enlarged to other affinity based separation or analytical systems, including affinity chromatography systems and the like, through no more than routine experimentation. The excinuclease protection assay is based directly on the steric hindrance, by bound SSRP, of DNA lesion repair by a eukaryotic DNA repair enzyme. In this assay, the lesioned DNA/SSRP complex is contacted with excinuclease and incubated therewith under conditions sufficient for the excinuclease-catalyzed removal of lesions from DNA. If a DNA lesion is accessible to the excinuclease, a single-stranded nucleic acid fragment comprising the lesion is removed from the double-stranded DNA. Typically, the fragment is less than 30 bp long. The resulting gap is filled with a patch of newly synthesized DNA complementary to the sequence of the unlesioned strand. Using appropriate nucleic acid labeling techniques, described herein, one or more of the nucleic acid products of successful excinuclease repair can be detected. Failure to excise a lesion from DNA, or the degree (e.g., percent) of inhibition thereof indicates SSRP shielding and thus is reasonably correlated with persistence of lesions in the genome.
To facilitate detection of lesioned DNA/SSRP lesions according to the foregoing methods, the invention also provides kits comprising, as applicable, one or more SSRPs, optionally formulated as a cocktail, probe DNA bearing a defined cisplatin-type lesion or in which such a lesion can be produced, a DNA labeling reagent, and optionally a detection or separation reagent selected from an excinuclease preparation and a nitrocellulose filter. Kit components are conveniently packaged for either manual or automated practice of the foregoing methods.
In still another aspect, the invention features a method of sensitizing eukaryotic cells to a genotoxic agent, including a method of rendering eukaryotic cells naturally resistant to cell killing by the genotoxic agent vulnerable thereto. Thus, this aspect of the invention features a method of enhancing cytotoxic effectiveness of a genotoxic agent that normally inflicts only transient lesions on cellular DNA. In this method, eukaryotic cells are contacted with nucleic acid encoding an SSRP that binds to genomic lesions produced by the genotoxic agent, under conditions sufficient for the nucleic acid to be internalized and expressed within said cells. The SSRP-encoding nucleic acid is a foreign (heterologous) nucleic acid, optionally a plasmid, cosmid, expression vector, or virus, e.g., a retrovirus. Intracellular expression of the encoded SSRP enhances persistence of genomic lesions, as the expressed SSRP shields lesions produced by the genotoxic agent from repair by cellular excinuclease. Nucleic acid encoding the SSRP can be caused to internalize within the cells by electroporation or microinjection. Alternatively, where the nucleic acid is present in an expression vector, it can be caused to internalize by transfection according to standard techniques or routine modifications thereof. Optionally, the internalized nucleic acid becomes integrated into the cellular genome. Preferably, the encoded SSRP is overexpressed within the cell, such that an excess of SSRP accumulates, thermodynamically favoring the formation of lesioned DNA/SSRP complexes at the sites of genomic lesions.
Accordingly, yet a further aspect of the invention features an improved method for killing eukaryotic cells. This improved method involves contacting the cells to be killed with nucleic acid encoding an SSRP that binds to lesions in DNA produced by a selected genotoxic agent, under conditions sufficient for the internalization and expression (preferably, overexpression) of the SSRP-encoding nucleic acid within the cells. The method further involves contacting the cells expressing the encoded SSRP with the selected genotoxic agent, under conditions sufficient for the formation of persistent and therefore cytotoxic lesions in the cell genome. Advantageously, then, the invention may allow the use of low doses of the genotoxic agent, formerly considered poorly effective or ineffective for cell killing. The invention also may enhance the effectiveness of additional genotoxins, including genotoxins formerly considered poorly effective or ineffective as cytotoxins. Further, the invention may reconstitute the cytotoxic susceptibility of cells that are refractory to killing by genotoxins, including cells that express a gene for multiple drug resistance.
Eukaryotic cells with which the foregoing methods can be practiced can be cells of a unicellular or multicellular organism. The cells can be maintained in or adapted to culture ex vivo, or can be cells withdrawn from a multicellular organism (e.g., a body fluid sample or tissue biopsy). Alternatively, the cells can be present in vivo in tissue or organs of a multicellular eukaryotic organism. The term, multicellular eukaryotic organism, embraces at least arthropods and vertebrates, including fish, amphibians, birds and mammals, particularly humans. The eukaryotic cells can exhibit either normal or transformed phenotypes. Thus, the eukaryotic cells can be transformed (neoplastic or malignant) cells, including carcinoma cells and sarcoma cells. Transformed mammalian cells with which the present invention can be practiced include transformed cells arising within any body tissue or body compartment, including transformed cells of central or peripheral nervous system, mammary, lymphoid, myeloid, cutaneous, respiratory tract, gastrointestinal tract, and urogenital tract origin. To assess susceptibility of transformed cells to killing by a desired chemotherapeutic genotoxin, a sample comprising the transformed cells can be withdrawn from an individual to be treated with the chemotherapeutic agent by standard biopsy techniques and processed for the release of intracellular proteins comprising endogenous SSRPs as described above. If desired, transformed cells can be sensitized to cell killing in situ by the genotoxic agent by causing them to internalize foreign nucleic acid encoding SSRP. Nucleic acid encoding SSRP can be administered to the individual using standard techniques or modifications thereof, appropriate to deliver the nucleic acid to the body compartment, organ or tissue harboring transformed cells. Preferably, the SSRP encoding nucleic acid is internalized by dividing cells, including transformed cells that have escaped normal physiologic and molecular restraints on cell proliferation and cell differentiation. Subsequent exposure of the SSRP-expressing transformed cells to a genotoxic agent according to accepted chemotherapeutic protocols or routine modifications thereof results in preferential killing in situ of the transformed cells.